Kirby-Bauer disk diffusion Testing skill based learning Dr.T.V.Rao MD Dr.T.V.Rao MD

Principles of susceptibility testing Dr.T.V.Rao MD

Kirby-Bauer Test is a agar disk diffusion method Kirby – Bauer Agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant bacterial isolates. Dr.T.V.Rao MD

Kirby-Bauer antibiotic testing Kirby-Bauer antibiotic testing (KB testing or disk diffusion antibiotic sensitivity testing) is a test which uses antibiotic-impregnated paper disks to test whether particular bacteria are susceptible to specific antibiotics. A known quantity of bacteria are grown on agar plates in the presence of thin filter paper discs containing relevant antibiotics. If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of inhibition). Dr.T.V.Rao MD

Agar disk diffusion method Medium Mueller Hinton 4 mm thickness pH 7.2 to 7.4 Antibiotic storage -20oC minimum disks temperature Inoculum McFarland 0.5 (108 bacteria/mL) Incubator temperature 35oC atmosphere ambient air 5 5

Procedure Prepare a pure culture (18-24 hrs) of the sample on a non- selective medium Adjust turbidity until it is equivalent to the 0.5 McFarland Turbidity Standard Use a Wickerham Card as background when adjusting the turbidity Sample 0.5 McFarland Standard Dr.T.V.Rao MD

Aseptic transfer of colonies for propagation of bacteria Select at least 4 to 5 well-isolated colonies of the same morphological type from an agar plate. Touch the top of each colony with a wire loop and transfer the growth to a tube containing 4 to 5 mL of a suitable broth medium, such as tryptic-soy broth. Allow the broth culture to incubate at 35°C until it achieves or exceeds the turbidity of 0.5 McFarland standard. Dr.T.V.Rao MD

Choosing a colony for testing Dr.T.V.Rao MD

McFarland 0.5 and Adjusted Test Organism Dr.T.V.Rao MD

Inoculate the plate with uniformity Within 15 minutes after adjusting the turbidity of the inoculum suspension, dip a sterile non-toxic swab on an applicator into the adjusted suspension. Rotate the swab several times, pressing firmly on the inside wall of the tube above the fluid level to remove excess inoculum from the swab. Dr.T.V.Rao MD

Uniform inoculum on plates is essential for effective detection Inoculate the dried surface of a Muller-Hinton agar plate by streaking the swab over the entire sterile agar surface. Repeat this procedure two more times, and rotate the plate 60° each time to ensure an even distribution of inoculum. Replace the plate top and allow 3 to 5 minutes, but no longer than 15 minutes, for any excess surface moisture to be absorbed before applying the antibiotic disks. Dr.T.V.Rao MD

Procedure Within 15 minutes of adjusting the turbidity dip a sterile cotton swab into the sample streak a lawn of bacteria on Mueller-Hinton agar Leave the lid agar for 3-5 minutes (no more than 15 minutes) to allow plate to dry Dr.T.V.Rao MD

Be cautious The agar medium should have pH 7.2 to 7.4 at room temperature.  The surface should be moist but without droplet of moisture. The antibiotic disks should be maintained at 8°C or lower or freeze at -14°C or below until needed, according to the manufacturer's recom­ mendations. Allow the disks to warm to room temperature before use. Don't use expired disks. Dr.T.V.Rao MD

Getting ideal results depend on right inoculum There should be an almost confluent lawn of growth when done properly. If only isolated colonies grow, the inoculum was too light and the test should be repeated. To avoid extremes in inoculum density, never use undiluted overnight broth cultures for streaking plates./li> Dr.T.V.Rao MD

Procedure Apply antibiotic impregnated disks on the bacterial lawn Important: where the disk drops is where it stays Incubate for 16-18 hours at 33 ± 2oC unless otherwise instructed Dr.T.V.Rao MD

Do not load the plates with too many antibiotic disks Place the appropriate disks evenly (no closer than 24 mm from center to center) on the surface of the agar plate either by using a sterile forceps or the dispensing apparatus. No more than 12 disks should be placed on one 150 mm plate or more than 5 disks on a 100 mm plate. A disk is not to be moved once it has come in contact with the agar surface since some of the compound diffuses almost instan­taneously. Dr.T.V.Rao MD

Proceed with incubation of plates Invert the plate and place them in an incubator at 35°C within 15 minutes after disks are applied. The plates should be incubated aerobically . After 16-18 hrs. of incubation, examine each plate and measure the diameters of the zones of complete   inhibition,   including   the diameter of the disk. Measure the zones to the nearest millimeter using a ruler. Large colonies growing within a clear zone of inhibition should be subcultured, reidentified and retested to know whether they are contaminants or mutants Dr.T.V.Rao MD

Interpretation c and C are the minor and major breakpoints The main concept is the “clinical categorisation" Strains are sorted according to level of Minimal Inhibitory Concentration (MIC) versus reference breakpoints c and C are the minor and major breakpoints Susceptible Intermediate Resistant MIC < c ≤ MIC < C ≤ MIC

Understanding breakpoints Words of laboratory specialists It is not possible to work alone Breakpoints are the expression of a consensus among the scientific community at a given time in a country Breakpoints are determined using two approaches Pharmacological concept Epidemiological concept

Results Antibiotics diffuse out onto the agar Concentration of antibiotics decrease as they diffuse further away from the disks After incubation, observe for a clearing on the bacterial lawn (zone of inhibition) Bacterial growth incubation Zone of inhibition Dr.T.V.Rao MD

Follow the guidelines for measuring zones of resistance and susceptibility Interpret the zone sizes by referring to the manufacturer provided standard table and report the organism to be  either susceptible, intermediate, or resistant. Never compare the zone sizes of two different antibiotics and judge their effectiveness accordingly. Dr.T.V.Rao MD

Results Measure the diameters of the zone of inhibition Interpret the results as “resistant” or “susceptible” according to the guideline provided by the CLSI Interpretation of the zone of inhibition is different for each bacteria-antibiotic combination Dr.T.V.Rao MD

Disk Susceptibility Testing Problems proper inoculum and heavy inoculum Dr.T.V.Rao MD 23 23

Disk Susceptibility Testing Problems disk properly applied disk not properly applied Dr.T.V.Rao MD

An agar gel that is too thick leads to smaller zones Dr.T.V.Rao MD

Standard strains for quality assurance Precision and accuracy ensured through control strains Known susceptibility to antimicrobial agents Standard strains include Staphylococcus aureus ATCC 25923 Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853

E-test® Same principle as the Kirby-Bauer Test Uses a plastic strip with a predefined gradient of antibiotic concentration Results are read directly on the strip where the zone of inhibition intersects with the strip E Zone of inhibition Bacterial growth Dr.T.V.Rao MD

Results Interpret results as “resistant” or “susceptible” according to the guidelines provided in the package insert For ambiguous results, refer to the provided reading guide for : Organism related effects Drug related effects Resistance mechanism related effects Technical and handling effects Dr.T.V.Rao MD

Patient results may be incorrect if: The organism was misidentified A clerical error was made Inappropriate choice of antimicrobials were tested and reported The wrong patient’s sample was examined The wrong test was ordered The sample was not preserved properly

Antimicrobial Susceptibility Testing Goals When possible, don’t duplicate systems Meet regulatory and other guiding requirements Use automation when possible and technically sound Have flexibility in antimicrobial battery Focus technologist time on tasks that utilize their training and expertise Get accurate results out as soon as possible Large CF population Disc diffusion quality control testing 65% agents concurrently being tested for quality assurance on existing Etest™ batteries Advances in Vitek allow for Dtest, cefoxiting and VRSA screen to be automated DO NOT COMPROMISE QUALITY FOR SPEED

Programme created by Dr. T. V Programme created by Dr.T.V.Rao MD for Microbiologists in the Developing World Email doctortvrao@gmail.com Dr.T.V.Rao MD